Folding Mechanisms of Dihydrofolate Reductase and the Response Regulators

本项目的目标是对生物学中最常见的α / β / α三明治基序的两个成员的折叠机制进行比较分析。先前nsf支持的对该基序的一个亚类——二氢叶酸还原酶（DHFR）折叠机制的研究表明，这种小的双结构域蛋白在四个平行通道中通过一系列通路上的部分折叠状态进行折叠。相比之下，黄伏氧素折叠家族的两个成员，该基序的另一个亚类，最初错误折叠成一个非通路中间体，必须至少部分展开才能进入导致天然构象的生产过渡状态。这种错误折叠反应的分子基础将通过实验和计算方法的结合来探索，这些方法将集中于确定黄伏氧毒素折叠的所有响应调节子类成员CheY， NtrC和Spo0F的这些中间体的结构特征和能量学。微秒折叠反应的连续流（CF）小角x射线散射（SAXS）、时间分辨福斯特共振能量转移（trFRET）和远紫外圆二色性（CD）测量将提供尺寸性质和全局二级结构的定量信息。脉冲猝灭氢交换方法和质量标记肽的质谱分析将通过识别定义稳定性核心的片段来增强圆二色性数据。突变分析将探索单个侧链在CheY折叠和错误折叠中的作用，特别关注中央β -薄片两侧疏水核中的大型非极性侧链。链熵在CheY错误折叠反应中的作用将通过在保持3D结构的同时创造改变链连通性的排列来测试。以响应调节因子折叠机制为中心的协作模拟将为错误折叠的物种提供详细的结构见解，以及它们回溯到原生构象的过程。与DHFR通路上亚毫秒折叠反应的结果进行比较，将为与反应调节因子的对比提供更多的见解。这种多维比较分析的结果有望增强对α / β / α三明治蛋白早期折叠反应的理解，并为黄氧还蛋白折叠中的错误折叠反应提供基础。这个项目的更广泛影响包括培训、教育和技术发展。本科生、研究生和博士后将接受分子生物物理学方面的培训，包括光谱方法、热力学和动力学、复杂数据分析、蛋白质工程和蛋白质折叠问题。所采用的几种方法，包括圆二色性和荧光光谱，以及开发的数据分析算法，将成为PI系研究生生物物理方法新高级主题课程的一部分。在蛋白质折叠机制的研究过程中开发的分析工具目前正在世界各地的十几个研究实验室中使用，并将通过生物化学和分子药理学部门赞助的网站提供给更多的人。在之前的拨款期间开发的连续流混合技术将继续优化，并通过合作（用于CF-trFRET和CF-CD）或通过阿贡国家实验室先进光子源的BioCAT光束线（用于CF-SAXS）提供给所有用户。

The goal of this project is to perform a comparative analysis of the folding mechanisms of two members of the alpha/beta/alpha sandwich motif, one of the most common in biology. Previous NSF-supported studies of the mechanism of folding of one sub-class of this motif, dihydrofolate reductase (DHFR), have shown that this small two-domain protein folds via a sequential set of on-pathway partially-folded states in four parallel channels. By contrast, two members of the flavodoxin fold family, another sub-class of this motif, initially misfold to an off-pathway intermediate that must at least partially unfold to access the productive transition state leading to the native conformation. The molecular basis for this misfolding reaction will be probed by a combination of experimental and computational methods that will focus on determining the structural features and energetics of these intermediates for CheY, NtrC and Spo0F, all members of the response regulator sub-class of the flavodoxin fold. Continuous-flow (CF) small-angle x-ray scattering (SAXS), time-resolved Forster resonance energy transfer (trFRET) and far-UV circular dichroism (CD) measurements of microsecond folding reactions will provide quantitative information on dimensional properties and global secondary structure. Pulse-quench hydrogen exchange methodology and mass spectrometric analysis of mass-labeled peptides will enhance the circular dichroism data by identifying the segments that define the cores of stability. Mutational analysis will explore the roles of individual side chains in the folding and misfolding of CheY, with a particular focus on large nonpolar side chains in the pair of hydrophobic cores on either side of the central beta-sheet. The role of chain entropy in the CheY misfolding reaction will be tested by creating permutations that vary the chain connectivity while preserving 3D structure. Collaborative native-centric simulations of response regulator folding mechanisms will provide detailed structural insights into the misfolded species and the process by which they backtrack to the native conformations. Comparisons with the results for the on-pathway sub-millisecond folding reaction in DHFR will provide additional insights into the contrast with response regulators. The results of this multi-dimensional comparative analysis are expected to enhance the understanding of early folding reactions in alpha/beta/alpha sandwich proteins and the basis for the misfolding reactions in the flavodoxin fold.The broader impact of this project has training, educational and technology development components. Undergraduate, graduate and postdoctoral fellows will receive training in molecular biophysics, including spectroscopic methods, thermodynamics and kinetics, sophisticated data analysis, protein engineering, and the protein folding problem. Several of the methods employed, including circular dichroism and fluorescence spectroscopy, and data analysis algorithms developed will form a part of a new advanced topics course in Biophysical Methods for graduate students in the PI's Department. The analytical tools developed during the course of this research on protein folding mechanisms are currently being used in over a dozen research labs around the world, and will be made more accessible via a website sponsored by the Biochemistry and Molecular Pharmacology Department. The continuous-flow mixing technology developed during the previous grant period will continue to be optimized and available to all users by collaboration (for CF-trFRET and CF-CD) or through the BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory (for CF-SAXS).